The inability to treat many Gram-negative bacterial infections effectively with existing antibiotics is a major medical crisis. Pseudomonas aeruginosa is a prime example: 30% of clinical isolates from critically ill patients are resistant to three or more drugs. The overall goal of this project is to address the critical medical need by a novel approach of identifying specific inhibitors of the type-three secretion system (T3SS) targeting the extracellular T3SS needle and developing them into novel therapeutic agents against P. aeruginosa. T3SS is the major virulence factor contributing to the establishment and dissemination of P. aeruginosa infections and is utilized by the bacterium to secrete and translocate toxin effectors into host phagocytes and weaken the host's innate immune response. The presence of a functional T3SS is significantly associated with poor clinical outcomes and death in patients and markedly reduces survival in animal infection models. T3SS inhibitors will be administered therapeutically and prophylactically in combination with anti-pseudomonal agents to inhibit the T3SS, potentiate a robust host innate immune response, and enhance the antibacterial activity of co-administered antibiotics. The strategy is to identify and optimize small molecules that interfere with the extracellular T3SS needle polymerization or stability. Such therapeutics will by-pass P. aeruginosa intrinsic resistance mechanisms caused by a poorly permeable outer membrane and efflux pumps. Preliminary studies revealed a putative binding site for the phenoxyacetamide series of T3SS inhibitors in the polymeric form of the needle protein PscF, indicating that the needle is a target of the P. aeruginosa T3SS for small molecule inhibition. The strategy of screening directly for compounds that alter the needle assembly or stability will capitalize on this newfound vulnerability and provide additional chemotypes of needle inhibitors for the drug development pipeline. In other preliminary studies, we developed methods for the purification of PscF and demonstrated a fluorescence-based assay to monitor the polymerization of a purified T3SS needle protein. In Phase I, a high-throughput screen using purified PscF will be developed, optimized and implemented to identify small molecules that inhibit needle polymerization or stability. Diverse compound libraries will be screened, and resulting 'hits' will be confirmed in the screening assay in replicate, prioritized b potency, and selectivity by eliminating compounds that alter actin polymerization or stability or are promiscuous in multiple screens. Confirmed potent, selective 'hits' will be validated as T3SS inhibitors by determining their ability to inhibit effector secretion and translocation from P. aeruginosa, and by ensuring that they are not cytotoxic, do not disrupt mammalian cell membranes, and do not affect bacterial growth or viability in vitro. Preliminary SAR and in vitro ADME assays and will be used to prioritize analogs. In Phase II, the most promising of these T3SS inhibitors will be optimized to develop lead compounds for efficacy and toxicity testing in animal models.